Do-it-yourself Gene Synthesis Kit

We have a variety of strains that are used in production, such as Top10 or DH5α™. Routinely, we grow them in dam+ strains. Therefore, you may be seeing inhibition because Xba1 is sensitive to dam methylation.

Overdigestion with CorrectASE™ enzyme can lead to degradation of the DNA template.

Ensure that the reaction does not go longer than 60 mins. Also, ensure that the reaction is kept on ice until the PCR step or else the reaction will be prone to overdigestion by the CorrectASE™ enzyme.

The protocol states that oligonucleotide stocks should be prepared at a final concentration of 100 μM in 1X TE buffer. The next line indicates the addition of 5 μL of each 10 μM primer together. According to R&D, the manual was written this way because our R&D typically brings up the lyophilized oligos to a 100 μM stock concentration (due to the volume of the tube). You do, however, want to use a 0.15 μM pool. Therefore, you can either dilute the stock to 10 μM or dilute the primer pool 1:10.

GeneArt® Site-Directed Mutagenesis Kits

What polymerase are you using? We recommend using AccuPrime™ Pfx DNA polymerase with both mutagenesis kits – use 1 unit of the polymerase for amplification.

Try optimizing the annealing temperature and extension time. The rule of thumb is 5-10 degrees C below your primer’s lowest melting temperature for the annealing temperature, and 30 seconds per 1kb for extension time. You can experiment with different temperatures/times to optimize your reaction.

Poor primer design could result in no product. We recommend using our tool to reduce potential secondary structures or increase the primer length.